MX2011002022A - The an3 protein complex and its use for plant growth promotion. - Google Patents

Abstract

The present invention relates to an AN3-based protein complex. It relates further to the use of the complex to promote plant growth, and to a method for stimulating the complex formation, by overexpressing at least two member of the complex.

Description

PROTEIN COMPLEX AN3 AND ITS USE TO PROMOTE THE VEGETABLE GROWTH The present invention relates to a protein complex based on A 3. It is also related to the use of the complex to promote plant growth, and with a method to stimulate complex formation, over-expressing at least two members of the complex .

The demand for more products derived from plants has increased dramatically. In the near future, the challenge for agriculture will be to meet the growth demands for forages and food in a sustainable manner. In addition, plants begin to play an important role as sources of energy. To face these great challenges, a profound increase in plant performance will have to be achieved. The production of biomass is a multifactorial system in which an excess of processes leads to the activity of meristems that give rise to new cells, tissues and organs. Although a considerable amount of research is being done on production performance, little is known about the molecular networks that underpin yield (Van Camp, 2005). Many genes have been described in Arabidopsis thaliana that, when mutated or expressed ectopically, result in the formation of large structures, such as leaves or roots.

These so-called "intrinsic performance genes" are involved in many different processes whose interrelation is almost unknown.

One of these "intrinsic performance genes", AN3 (also known as GIF1), was identified in the search for GRF (growth regulation factor) interactors (Kim and Kende, 2004) and by analysis of leaf Arabidopsis mutants. narrow (Horiguchi et al., 2005). AN3 is a homolog of the human SYT protein (growth regulatory shift) and is encoded by a small genetic family in the Arabidopsis genome. SYT is a co-activator of transcription whose biological function, despite the implication of its chromosomal shift in carcinogenesis is still uncertain (Clark et al., 1994, de Brujin et al., 1996). When using the GAL 4 yeast system, A 3 showed that it has a transactivation activity (Kim and Kende, 2004). This, together with the double hybrid yeast and in vitro binding assays demonstrating the interaction of A 3 with several GRFs (Kim and Kende, 2004; Horiguchi et al., 2005), suggests a role of A 3 as the co-activator of transcription of GRFs. The GRF (growth factor) genes take place in the genomes of all plants that produce seeds, and until now they have been presumed to encode transcription factors that play a regulatory role in the growth and development of leaves (Kim et al., 2003). In support of a transcription activator of GRF and A 3 and a co-activating complex, the grf and an3 mutants display similar phenotypes, and combinations of grf and an3 mutations demonstrated a cooperative effect (Kim and ende, 2004). It was shown that the an3 mutant leaf narrow phenotype results from a reduction in cell numbers. In addition, the 'ectopic expression of AN3 results in transgenic plants with larger leaves consisting of more cells, indicating that AN3 controls both cell number and organ size (Horiguchi et al., 2005). Although the function of AN3 in the regulation of plant growth is not known, these results show that ?? 3 meets the requirements of an "intrinsic yield gene".

In our effort to decipher the molecular network by supporting a mechanism to improve performance, a focused procedure of broad genome proteins was carried out to study A 3 interactive proteins in cell suspension cultures of Arabidopsis thaliana. The technology of affinity purification in tandem (TAP) combined with a protein identification based on mass spectrometry (MS) results in the isolation and identification of 25 interactive proteins of AN3 that can act in the regulation of plant growth (Table 2 ). Surprisingly, several proteins that are part of multiple protein complexes were isolated. In addition, many interactors are not fully characterized. Reports on some of the AN3 interactors show that they are involved in various evolutionary processes (Wagner & amp;; Meyerowitz, 2002; Meagher et al., 2005; Sarnowski et al., 2005; Hurtado et al., 2006; Kwon et al., 2006) although to date none of the identified genes have been associated with the stimulation of plant growth.

A first aspect of the invention is a complex of proteins based on isolated AN3, comprising at least the AN3p proteins and one or more proteins selected from the group encoded by AT4G16143, AT1G09270, AT3G06720, AT5G53480, AT3G60830, AT1G18450, AT2G46020, AT2G28290, ATIG21700, AT5G14170, AT4G17330, AT4G27550, AT1G65980, AT5G55210, AT3G15000, AT4G35550, AT1G20670, AT1G08730, AT5G13030, AT2G18876, AT5G17510, AT1G05370, AT4G21540, AT1G23900 and AT5G23690 (genes listed in Table II). Preferably, the protein complex based on A 3 > it comprises at least 3p A proteins and one or more proteins selected from the group consisting of ARP4 (AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD (AT2G28290), SWI3C (AT1G21700) and SWP73B (AT5G14170).

Even more preferably, the A 3 -based protein complex comprises at least A 3p, an actin-related protein, selected from the group consisting of ARP4 and ARP7, an ATPase selected from the group consisting of SNF2 (BRM) and SYD and a protein that contains the S IRM domain. Preferably, the protein containing the SWIRM domain is SWI3C. A protein complex based on AN3 as used herein means that the AN3p is interacting, directly or indirectly, with the other proteins of the complex. A direct interaction is an interaction where at least one AN3p domain interacts with one or more domains or the interactive partner. A direct interaction is an interaction where the AN3p itself is not interacting with the interactive protein through one of its domains, although the interactive protein is interacting with a protein that is directly or indirectly interacting with AN3p.

A further aspect of the invention is the use of overexpression of the protein complex, by over-expressing at least two members of the protein complex. The promotion of plant growth, as used herein, is an increase in plant biomass in plants where the protein complex is used, as compared to the same plant where the complex is not used, developed under the same conditions , except for the conditions necessary for the use of the complex, if there is one. Such conditions may be, as an example not limited, the addition of one or more compounds to induce one or more promoters of one or more genes encoding a protein of the complex. Alternatively, the same plant is a non-transformed parent plant, developed under the same conditions as the transformed plant, where the complex is used. Preferably, the promotion of plant growth results in increased yield. This yield may be a total increase in plant biomass, or a partial increase in yield, such as, but not limited to, seed yield, leaf yield or root yield.

In addition, another aspect of the invention is a method for promoting the formation of the protein complex based on A 3, by simultaneous overexpression of at least two proteins of the complex. The proteins of the complex, together with the AN3p itself, are listed in Table II. Preferably, overexpression is an overexpression of AN3p and one or more proteins selected from the group consisting of ARP4 (AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD (AT2G28290), SWI3C (AT1G21700) and SWP73B (AT5G14170) . Even more preferably, overexpression is an overexpression of at least A 3p, an actin-related protein selected from the group consisting of ARP4 and ARP7, an ATPase selected from the group consisting of SNF2 (BRM) and SYD and a protein which contains the SWIRM domain. Preferably, the protein containing the SWIRM domain is SWI3C.

Methods for obtaining overexpression are known to the person skilled in the art, and include, but are not limited to, placing the gene that encodes the protein that is overexpressed after a strong promoter such as the promoter of the 35S Virus. Mosaic of the cauliflower. The simultaneous overexpression as used herein means that there is an overlap in the term for all proteins that are overexpressed, so that the level of such proteins increases when compared to a control not over-expressed. It does not necessarily mean that all genes must be induced at the same time. Depending on the production of the messenger RNA and / or the protein, one gene can be induced before or after another, as long as there is an overlap in time where both proteins are present in a concentration that is higher than the normal concentration ( not over-expressed).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Analysis of expression of GFP tagged with GS and AN3 in transgenic cell suspension cultures.

The total 2-day natural protein extract and cultures over-expressing the GFP labeled with GS and AN3 N and C terminals (60 g) were separated by 12% SDS-PAGE and immuno-transferred. For detection of GS-tagged proteins, blots were incubated with human blood plasma followed by incubation with horseradish peroxidase-coupled anti-human IgG. Protein gel transfers were developed by chemiluminescent detection. The expected recombinant molecular masses for GFP labeled with GS and AN3 are 52.8 kDa and 43.5 kDa, respectively (indicated with a black dot).

Figure 2. Analysis of TAP protein eluates.

Protein complexes labeled with GS were purified from cultures of transgenic plant cell suspension, precipitated with TCA (25% v / v), separated in 4-12% of NuPAGE gels, and visualized with colloidal Coomassie G-250 staining. . Bait proteins are indicated with a point.

EXAMPLES Materials and methods for the examples Construction of the Vector The construction of GFP and AN3 labeled with GS N and C terminals under the control of the 35S promoter. { CaMV) was obtained through Multisite Gateway LR reactions. The coding regions without (-) and with (+) stop codon were amplified by polymerase chain reaction (PCR) and cloned into the Gateway vector pDONR221 (Invitrogen) resulting in pEntryLlL2-GFP (-), pEntryLlL2-GFP (+), pEntryLlL2-AN3 (-) and pEntryLlL2-A 3 (+). Plant transformation vectors containing Pro35s: GFP-GS- and Pro35s: AN3-GS were obtained by ultisite Gateway LR reaction between pEntryL4Rl-Pro35s, pEntryLlL2 -GFP (-) or pEntryLlL2-AN3 (-), and pEntryR2L3-GS and the target vector pKCTAP, respectively (Van Leene et al., 2007). To obtain the Pro35S vectors: GS-GFP and Pro35S: GS-AN3, the Multisite LR recombination takes place between pEntryL4L3-Pro35S and pEntryLlL2-GFP (+) or pEntryLlL2 -AN3 (+) with pKNGSTAP.

All access and destination vectors were examined by sequence analysis. The expression vectors were transformed to the strain Agrobac erium tumefaciens C58ClRifR (pMP90) by electroporation. Bacteria transformed into yeast extract plates containing 100 μg / mL of rifampin, 40 μg / mL of gentamicin and 100 μg / mL of spectinomycin were selected.

Cell suspension culture The natural and transgenic Arabidopsis thaliana cell suspension PSB-D cultures were maintained in 50 ml of the MSMO medium (4.43 g / L of MSMO, Sigma-Aldrich), 30 g / L of sucrose, 0.5 mg / L of NAA, 0.05 mg / L of kinetin, pH 5.7 adjusted with 1 KOH) at 25 ° C in the dark, by gentle agitation (130 rpm). Every 7 days, the cells were sub-cultured in a fresh medium in a 1/10 dilution.

Cell culture transformation The Arabidopsis culture was transformed by co-cultivation of Agrobacterium as previously described (Van Leene et al., 2007). The cultivation of Agro £ > Acteriu / r exponentially growing in YEB (OD60o between 1.0 and 1.5) was washed three times by centrifugation (10 minutes at 5000 rpm) with an equal volume of MSMO medium and resuspended in a cell suspension growth medium until an OD6oo of 1.0. Two days after the subculture, the suspension culture of 3 ml was incubated with 200 uL of washed Agrojbacteria and 200 μl. of acetosingone, for 48 hours in the dark at 25 ° C with gentle shaking (130 rpm). Two days after co-culture, 7 ml of MSMO containing a mixture of three antibiotics (25 μg / ml kanamycin, 500 μg / ml carbenicelline and 500 μg / ml vancomycin) were added to the cell cultures and cultured also in suspension under standard conditions (25 ° C, 130 rpm and continuous darkness). Stable transgenic cultures were selected by sequential dilution in a ratio of 1: 5 and 1:10 in 50 mL of fresh MSMO medium containing the antibiotic mixture, respectively at 11 and 18 days after co-culture. After counter- V To select the bacteria, the transgenic plant cells were further subcultured weekly in a 1: 5 ratio in 50 mL of the MSMO medium containing 25 g / mL of kanamycin for two more weeks. Subsequently, the cells were subcultured weekly in a fresh medium in a 1/10 dilution.

Expression analysis of cell suspension cultures Transgenic expression was analyzed in a total protein extract derived from exponentially growing cells, harvested two days after subculture. Equal amounts of total protein were separated on 12% SDS-PAGE gels and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). Transfers of protein gels in 3% skim milk were blocked in 20 mM Tris-HC1, pH 7.4, 150 mM NaCl and 0.1% Triton X-100. For detection of GS-tagged proteins, blots were incubated with human blood plasma followed by incubation with horseradish peroxidase-coupled anti-human IgG (HRP, Ge-Healthcare). Protein gel transfer was developed by chemiluminescent detection (Perkin Elmer, Norwalk, CT).

Preparation of protein extract Cell material (15 g) was ground for homogeneity in liquid nitrogen. Unpurified protein extracts were prepared in an equal volume (w / v) of extraction buffer (25 mM Tris-HCl, pH 7.6, 15 mM MgCl 2, 5 mM EGTA, 150 mM NaCl, 15 mM p -nitrophenyl phosphate, 60 mM of β-glycerophosphate, 0.1% (v / v) of Nonidet P-40 (NP-40), 0.1 mM of sodium vanadate, 1 mM of NaF, 1 mM of DTT, 1 mM of PMSF, 10 ug / mL of leupeptin, 10 pg / mL of soybean trypsin inhibitor, 0.1 mM of benzamidine, 1 μ of tradis-epoxysuccinyl-L-leucylamido- (4-guanidino) butane (E64), 5% ( v / v) of ethylene glycol) using an Ultra-Turrax T25 mixer (IKA Works, Wilmington, NC) at 4 ° C. The soluble protein fraction was obtained by a double-step centrifugation at 36,900 g for 20 minutes and at 178,000 g for 45 minutes at 4 ° C. The extract was passed through a 0.45 μm filter (Alltech, Deerfield, IL) and the protein content was determined with the Protein Assay kit (Bio-Rad, Hercules, CA).

Tandem affinity purification Purifications were performed as described by Bürckstümmer et al. (2006), with some modifications. Briefly, 200 mg of total protein extract were incubated for 1 hour at 4 ° C under gentle rotation with 100 uL of Sepharose 6 Fast Flow Flow IgG beads (GE-Healthcare, Little Chalfon, UK), pre-equilibrated with 3 mL of extraction buffer. The Sepharose IgG beads were transferred to a Mobicol column of 1 mL (MoBiTec, Goettingen, Germany) and washed with 10 mL of wash buffer (10 mM Tris-HCl, pH 8.0, 150 mL of NaCl, 0.1% NP-40, 5% ethylene glycol) and 5 mL of Tobacco Etch Virus buffer. { Nicotiana tabacum L.) (TEV) (10 mM Tris-HCl, pH 8.0, 150 ml NaCl, 0.1% (v / v) NP-40, 0.5 mM EDTA, 1 mM PMSF, 1 μm E64, 5% (v / v) of ethylene glycol). The binding complexes were eluted by AcTEV digestion (2 x 100U, Invitrogen) for 1 hour at 16 ° C. The fraction eluted with IgG was incubated for 1 hour at 4 ° C under gentle rotation with 100 μl of streptavidin resin (Stratagene, La Jolla, CA), pre-equilibrated with 3 ml of TEV buffer. Streptavidin beads were stored on a Mobicol column, and washed with 10 ml of TEV buffer. Binding complexes were eluted with 1 ml streptavidin elution buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (v / v) NP-40, 0.5 mM EDTA, 1 mM PMSF, 1 μ? Of E64, 5% (v / v) of ethylene glycol, 20 mM of Destiobiotin) and were precipitated using TCA (25% v / v). The protein granules were washed twice with ice-cold acetone containing 50 mM HC1, redissolved in sample buffer and separated in 4-12% gradient NuPAGE gels (Invitrogen). The proteins were visualized with colloidal Coomassie brilliant blue staining. Proteolysis and peptide isolation After decolorization, gel plates were washed for 1 hour in H20, the polypeptide disulfide bridges were reduced for 40 minutes in 25 mL of 6.66 mM DTT in 50 mM NH4HC03 and sequentially the thiol groups were alkylated for 30 minutes in 25 mL of 55 mM AMI in 50 mM NH4HCO3. After washing the gel plates 3 times with water, whole channels from the protein gels were cut into slices, harvested onto microtiter plates and treated essentially as described above with minor modifications (Van Leene et al., 2007). Using microtiter plate well, dehydrated gel particles were rehydrated in 20 μl of digestion buffer containing 250 ng of trypsin (MS Gold, Promega, Madison, wi), 50 mM of NH4HCO3 and 10% of CH3CN (v / v) for 30 minutes at 4 ° C. After adding 10 uL of a buffer containing 50 mM NH4HC03 and 10% CH3CN (v / v), proteins were digested at 37 ° C for 3 hours. The resulting peptides were concentrated and desalted with solid phase pipette tips in microcolumna (PerfectPureTM C18 pipette tip, 200 nL bed volume, Eppendorf, Hamburg, Germany) and eluted directly onto a MALDI contact plate (Opti-TOF ™ 384 Well Insert, Applied Biosystems, Foster City, CA) using 1.2 μL of 50% CH3CN solution: 0.1% CF3OOH saturated with a-cyano-4-hydroxycinnamic acid and enriched with 20 fmoles / uL of Glul-Fibrinopeptide B (Sigma-Aldrich), 20 fmol / L of des-Pro2-Bradykinin (Sigma-Aldrich), and 20 fmol / uL of the Human Adrenocorticotropic Hormone Fragment 18-39 (Sigma-Aldrich).

Acquisition of mass spectrum A MALDI contact MS instrument (4800 Proteomics Analyzer; Applied Biosystems) was used to acquire peptide mass mappings and the subsequent 1 kV CID fragmentation spectrum of selected peptides. The peptide mass spectrum and the spectrum of peptide sequences were obtained using parameters as presented essentially in Van Leene et al. (2007). Each MALDI plate was calibrated according to the manufacturer's specifications. All peptide mass mapping (PMF) spectra were initially calibrated with three internal standards m / z 963,516 (des-Pro2-Bradykinin), m / z 1570.677 (Glul-Fibrinopeptide B), and m / z 2465.198 (Fragment of Adrenocorticotropic Hormone 18-39) resulting in an average mass accuracy of 5 ppm ± 10 ppm for each peptide concentration analyzed in MALDI contact plates analyzed. When using the PMF individual spectrum, up to sixteen peptides were subjected, exceeding a signal to noise ratio of 20 passing through a mass exclusion filter for fragmentation analysis.

Identification of MS-based protein homology The PMF spectrum and the spectrum of peptide sequences of each sample were processed using the integrated program package (GPS Explorer 3.6, Applied Biosystems) with parameter settings essentially as described in Van Leene et al. (2007). Data search files were generated and presented for identification of protein homology using a local database search engine (ascot 2.1, Matrix Science) · A non-redundant internal Arabidopsis protein database called SNAPS Arabidopsis Thaliana version 0.4 (SNAPS = Simple Nonredundant Assembly of Protein Sequences, accesses of sequences 77488, residues 30468560, available at http://www.ptools.ua.ac.be/snaps) was compiled from nine public databases. Protein homology identifications of the main (first category) were retained with a relative score that exceeded 95% probability. Additional positive identifications (second category and more) were retained when the score exceeded 98% of the probability threshold.

EXAMPLE 1: Analysis of expression of cell lines that over-express GFP and AN3 labeled with GS.

Prior to performing TAP purifications, suspension cultures of stably transformed cells were selected at the level of expression of transgene proteins. The transfer of protein gel of equal amounts of protein extract derived from natural cultures (PSB-D) and cell lines that overexpress GS-GFP, GFP-GS, GS-AN3 and A 3-GS show a clear expression, of the proteins labeled with GS (Figure 1).

EXAMPLE 2: TAP purification of cultures that over-express natural GFP and labeled with GS.

Despite the two successive purification steps performed within TAP purifications, background proteins co-purified by non-specific binding are a problem. The contaminating proteins were determined due to the experimental background by purification in natural and transgenic cultures that overexpress a green localized fluorescence protein (GFP) labeled with GS N and C terminal. Non-specific co-purified proteins, gel separated, were stained (Figure 2), digested with trypsin and clearly identified by MALDI-TOF / TOF. Most contaminants are rich in proteins, such as chaperones, cytoskeletal proteins, ribosomal proteins, metabolic enzymes or protein translation factors (Table 1). Identical or similar proteins were found as common contaminants in other interactive protein-protein studies (Rohila et al., 2006; Van Leene et al., 2007).

EXAMPLE 3: Isolation of TAP and identification of MS of interactive proteins AN3.

In order to identify the interactive pairs of A 3 in vivo, tandem affinity purifications (TAP) were performed in GS 3 and C terminal fusions of A 3 ectopically expressed under the control of the constitutive 35SCaMV promoter in transgenic Arabidopsis suspension cultures. Two independent TAP purifications were performed in extracts of the AN3-GS and GS-AN3 lines, harvested two days after sub-cultivation in a fresh medium. Affinity-purified proteins were separated on a NuPAGE gel of 4-12% and stained with Coomassie Brilliant Blue. Shown in Figure 2 is the purification profile of transgenic cultures that overexpress A 3. Protein bands are cut in trypsin digested gel and subjected to MALDI-TOF / TOF mass spectrometry for protein identification. After subtracting the background proteins, identified by control purifications described in example 2 and in another analysis (GUS and cytosolic GFP, Van Leene et al., 2007) from the list of results, 25 interactive proteins of AN3 were identified (Table 2). These can be divided into two groups: 14 proteins were confirmed experimentally and 11 proteins were identified in only one of four TAP experiments.

EXAMPLE 4: Isolation and identification of complex SW1 / SNF chromatin remodeling complex subunits with AN3 in plants.

Among experimentally confirmed A 3 interactors, six proteins act as subunits of macromolecular machines, which remodel the structure of chromatin. A database survey (ChromDB, GEndler et al., 2008) illustrates that all belong to the SWI / SNF ATPase family, ATPases of chromatin SWI / SNF remodeling are conserved in the animal and the plant kingdom and regulate the programs of transcription in response to endogenous and exogenous signals. This suggests that the transcription activity of AN3 is regulated by chromatin remodeling. In concordance, the homologous SYT of human AN3 also showed that it interacts with the BRM and Brgl components of the SWI / SNF complex (Thaete et al., 1999, Perani et al., 2003, Ishida et al., 2004).

Although the functional role of several components of the putative SWI / SNF complex in Arabidopsis has been studied, until now no complete plant chromatin remodeling complex has been isolated and characterized. The co-purification with AN3 gives for the first time a test of the in vivo physical composition of SWI / SNF plant complexes which previously were based solely on homology analysis and the interpretation of genetic and in vitro interactions. A literature review illustrates which ATPase SWI / SNF subunits control multiple development trajectories in Arabidopsis. Null mutants of the two SYD (At2g28290) and BRM (SNF2) (At2g46020) Isolated ATPases display pleiotropic developmental defects. Both mutants are slow-growing and are extremely small, have defects in the separation of cotyledons and exhibit a reduced apical domain (agner &; Meyerowitz, 2002; Farrona et al., 2004; Hurtado et al., 2006; Kwon et al., 2006; Su et al., 2006). The null mutants in BRM (SNF2) also have unique root growth defects and are male sterile (Wagner &Meyerowitz, 2002; Hurtado et al., 2006; Kwon et al., 2006). Mutants of the basic Swi3c complex (Atlg21700) closely resemble brm mutants (Sarnowski et al., 2005). Mutants of the ARP4 and ARP7 helper components display pleiotropic defects with less affinity to the syd, brm and swi3c phenotypes (Meagher et al., 2005). The deregulation of ARP4 results in phenotypes including the altered organization of plant organs, early flowering, delayed aging of the flowers and partial sterility (Kandasamy et al., 2005a). The modification of ARP7 results in extremely small plants with small leaves in the form of roses, highly retarded root growth, altered flower development and reduced fertility (Kandasamy et al., 2005b). Finally, RNAi-mediated silencing of the S I / SNF auxiliary component SWP73B (At5gl4170) resulted in extremely small plants with shorter roots (Crane &Gelvin, 2007).

EXAMPLE 5: Isolation and identification of interactors of AN3 With the exception of the subunits of the SWI / SNF chromatin remodeling complex, all other identified AN3 interactors are not characterized or characterized poorly. Table 3 provides an overview of the biological process GO and molecular function.

Among these four interactors (At4gl6143, Atlg09270, At3g06720 and At5g53480) are involved in the nucleo-cytoplasmic traffic which identifies A 3 as one of the targets of the nuclear transporters of the plant. In fact, a precise cellular localization is essential for protein function and nuclear localization is a key to the function of transcription factors. In plants, nucleo-cytoplasmic traffic plays a critical role in various biological processes (Meier, 2007; Xu &Meier, 2008) and nuclear transporters have shown that they are involved in regulating different signal transduction trajectories during plant development (Bollman et al. al., 2003) and in responses of the plant to biotic (Palma et al., 2005) and abiotic stresses (Verslues et al., 2006).

Another interactor AN3, which is not yet characterized, is trehalose phosphatase / synthase 4 (TPS4). Several studies in plants imply an important role of trehalose biosynthesis for plant growth, development and tolerance to stress (Grennan, 2007). In the case of Arabidopsis TPS1, genetically modified mutants display an embryonic lethal phenotype, suggesting a role for this gene in plant development (Eastmond et al., 2002). In addition, the overexpression of TPS1 requires its role as a regulator of glucose, abscisic acid and voltage signaling (Avonce et al., 2004). The latest study, together with a recent analysis of a TPS of rice by triggering a genetic induction of abiotic stress response when over-expressed (Ge et al., 2008), suggests a possible role for TPS genes to regulate signaling pathways of transcription.

The other identified interactors indicate links of the AN3 function in multiple processes. Several studies demonstrate the involvement of sphingosine kinases in plant cell signaling (Coursol et al., 2003; Coursol et al., 2005; Worral et al., 2008), while reports in myosin homologs (Peremyslov et al., 2008; Jiang et al., 2007) involve protein trafficking roles and organelles in plant development. The connections between these genes, the other interactors identified? AN3 will be interesting to study in the future.

Table 1. List of co-purifying proteins during TAP experiments of untransformed cell cultures, and of cultures that ectopically express a nuclear localized GFP Registration number Protein name Imitation GFP At1g06780 + glycosyltransferase protein of family 8 At1g07930 factor 1 -alpha lengthening + At1g09080 luminal binding protein 3 (BIP-3) (BP3) + At1g13440 glyceraldedo 3-phosphate dehydrogenase. cytosolic + At1g31230 bifunctional asparatate kinase / homoserine dehydrogenase + At1g34610 protein from the Ulp1 protease + family At1g50010 alpha tubulin + chain At1g61210 repeat family protein WD-40 / p80 subunit + katanina, supposed At1g75010 protein containing MORN + repeat At1g79920 heat shock protein 70, assumed + At1g79930 heat shock protein, putative + At2g07620 supposed helicase + At2g21410 Vacuolar proton ATPase, putative + At2g26570 expressed protein + At3g07160 protein glycosyltransferase of the family 48 + At3g09170 protein from the Ulpl protease family + At3g09440 70 kDa of protein 3 of the thermal shock cognate + At3g1 1950 ATHST; prenyltransferase + At3g12580 heat shock protein 70, assumed + At3g17390 S-adenosylmetlonine synthetase, supposed + At3g18530 protein expressed + At3g26020 subunit B 'regulatory protein phosphatase 2A + serine / threonine At3g42100 related protein that contains an AT + hook motif At3g48870 ClP + Protein ATP Binding Subunit (CIPC) dependent on ATP At3g49640 protein family nitrogen regulation + At3g54940 cysteine proteinase, putative + At4g00020 BRCA2A (breast cancer 2 as 2A) + At4g09800 S18 ribosomal protein 40S + At4g14960 alpha + tubulin chain At4g18080 hypothetical protein + At4g20160 expressed protein + At4g20890 beta + tubulin chain At4g31820 Protein of the NPH3 sensitive phototropic family + At4g33200 myosin, supposed. + At5g02490 70 kDa of heat shock cognate protein + 2 At5g0250O 70 kDa of heat shock cognate protein + At5g08670 ATP beta synthase chain, myotondrial + AtSg08680 ATP beta synthase chain, myotondrial + At5g08690 ATP beta synthase chain, myotondrial + At5g09810 actin (ACT7) / actin 2 ++ A (5g181 10 novel coat binding protein (nCBP) + At5g28540 luminal binding protein 1 (BiP-1) (BP1) + + + At5g35360 acetyl-CoA carboxylase, biotin carboxylase subunit (CAC2) At5g40060 disease resistance protein (class TIR-NBS-LRR), + supposed At5g42020 luminal binding protein 2 (BiP-2) (BP2) + At5g44340 beta + tubulin chain At5g60390 factor 1 -alpha lengthening + At5g62700 beta + tubulin chain Table 2. List of proteins co-purified with AM3 identified by MS. The last column tells how many of the four independent experiments were identified as interactor.

Count Score / Score / AGI Code Description MW of threshold threshold of peptide proteins optimal ion (kDa) AT4G16143 49.5 13 388/61 - 84/28 2 alleged alpha-2 importin (IMPA2) 1 AT1G09270 subunit of importin alpha-1, assumed (I PA4) 59.4 6 74/61 37/31 2 AT3G06720 subunit of importin alpha-1, assumed (IMPA1) 58.6 8 160/61 62/28 2 AT5G53480 importin beta-2. supposed 96.2 16 295/61 50/32 3 AT3G60830 protein 7 related to actin (ARP7) 39.9 12 285/61 53/28 2 AT1G18450 protein 4 related to actin (ARP4) 48.9 12 230/61 44/28 2 AT2G46020 Transcription regulatory SNF2 protein 245.4 31 351/61 57/31 (ATPase) 4 AT2G28290 chromatin remodeling protein, SYD 389.8 22 118/61 53/31 ATPase 2 AT1 G21700 protein that contains the domain 88.2 5 32/32 SWIRM / protein binding family of DNA AT5G14170 protein that contains the BAF60b domain of 59.2 18 302/6 43/31 comoleio SWIB AT4G17330 G2484-1, protein containing the agent domain 113.3 25 317/61 61/32 (as tudor) 2 AT4G27550 trehalose phosphatases / synthase 4 89.4 15 68/61 2 AT1 G65980 thioredoxin-dependent peroxidase 17.4 8 80/61 2 AT5G55210 protein expressed 18.5 4 105/61 49/31 2 AT3G15000 protein expressed similar to protein DAG 42.8 3 38/30 AT4G35550 homeo-leucine box zipper protein (HB- 29.6 3 33/28 2V HD-ZIP Protein AT1 G20670 protein containing bromodomain binding 72.9 16 75/61 DNA AT1G08730 myosin heavy chain (PCR43) (Fragment) 174.6 18 70/61 AT5G13030 protein expressed 71, 1 3 31/29 1 AT2G18876 protein expressed 43.5 1 1 67/61 1 AT5G17510 protein expressed 42.5 3 37/28 AT1G05370 protein expressed 49.9 12 66/61 1 AT4G21540 Assumed cystase sphingosine (SphK) 141.7 9 69/61 1 AT1G23900 gamma-adaptin 96.4 19 78/61 1 AT5G23690 protein of the polynucleotide family 59.6 11 66/61 adenyltransferase 1 Table 3 Code Name / Description Biological Process GO Molecular Function GO AGI At4g16143 Importina alfa-2 (IMP2) Imported protein inside Core protein transporter activity At1 g09270 Importina alfa-1 (I PA4) Protein transport Activity transportadora de Ncelcellular oroteins At3g06720 Importina alfa-1 (IMPA1) Protein transport Protein activity of intracellular proteins At5g53480 Importina beta-2 Imported protein inside Core protein transporter activity At4g17330 Protein G2484-1 Unknown RNA binding At4g27550 Trehalose phosphatase / synthase 4 Trehalose biosynthesis Trehalose activity (TPS4) phosphate synthase At1 g65980 Peroxidase 1 dependent on Unknown Antioxidant activity NnrfiHnxina GG ??? At5g55210 Protein expressed Unknown Unknown At3g15000 Expressed protein similar to Unknown Unknown DAG protein At4g35550 Baking box 13 related to Transcription regulation DNA binding Wuschel ?? / ?? 13} At1 g20670 Protein containing bromodomain Unknown DNA binding At1 g08730 XIC protein similar to myosin Movement based on protein binding filament At5g 13030 Protein expressed Unknown Unknown At2g 18876 Protein expressed Unknown Unknown At5g17510 Protein expressed Unknown Unknown At1 g05370 Protein expressed Unknown Unknown At4g21540 Assumed Sphingosine Kinase Activity Activation of Kinase Activity protein kinase C At1g23900 Gamma-adaptin Transport mediated by clathrin binding At5g23690 Polynucleotide protein RNA processing RNA binding REFERENCES Avonce N, Leyman B, Mascorro-Gallardo JO, Van Dijck P, Thevelein JM, Iturriaga G (2004) The Arabidopsis trehalose-6-? synthase AtTPSl gene is a regulator of glucose, abscisic acid, and stress signaling. 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Claims (6)

1. A protein complex based on isolated A 3, comprising at least the AN3p proteins and one or more of the proteins selected from the group encoded by AT4G16143, AT1G09270, AT3G06720, AT5G53480, AT3G60830, AT1G18450, AT2G46020, AT2G28290, ATIG21700, AT5G14170, AT4G17330, AT4G27550, AT1G65980, AT5G55210, AT3G15000, AT4G35550, AT1G20670, AT1G08730, AT5G13030, AT2G18876, AT5G17510, AT1G05370, AT4G21540, AT1G23900 and AT5G23690

2. A protein complex based on isolated AN3 comprises at least 3p A proteins and one or more proteins selected from the group consisting of ARP4 (AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD (AT2G28290), SWI3C (AT1G21700) and SWP73B (AT5G14170).

3. An isolated A 3 -based protein complex according to claim 2, whereby the protein complex comprises at least AN3p, an actin-related protein selected from the group consisting of ARP4 and ARP7, an ATPase selected from the group consists of SNF2 (BRM) and SYD and a protein that contains the SWIRM domain.

4. An isolated A 3 -based protein complex according to claim 3, whereby the protein containing the SWIRM domain is SWI3C.

. The use of a protein complex according to any of the preceding claims to promote plant growth.

6. A method for promoting the formation of the protein complex based on A 3 by simultaneous overexpression of at least two proteins of the complex.